Process for the enzymatic resolution of 2-amino-4-methyl-phosphinobutyric acid derivatives

ABSTRACT

L-PTC (L-phosphinothricin, L-2-amino-4-methylphosphino-butyric acid) is the active herbicidal component of D,L-PTC and can be obtained according to the invention when D,L-PTC derivatives which are N-acylated and esterified on the phosphinic acid group as well as optionally esterified or amidated on the carboxylic group, are treated with a hydrolytically active enzyme in an aqueous or aqueous-organic medium, in which process the L-PTC derivatives are selectively hydrolyzed on the N-acyl group or the modified carboxyl group, the resulting product mixture is resolved, and the desired L-PTC derivative is hydrolyzed to give the L-PTC and isolated by customary methods.

This application is a Divisional of prior application Ser. No.08/589,999, filed Jan. 23, 1996, now U.S. Pat. No. 5,756,346, which inturn is a continuation of application Ser. No. 08/324,536 filed on Oct.18, 1994, now abandoned, which in turn is a continuation of applicationSer. No. 08/182,668, filed on Jan. 14, 1994, now abandoned, which inturn is a continuation of application Ser. No. 08/018,460, filed on Feb.16, 1993, now abandoned, which in turn is a continuation of applicationSer. No. 07/474,499, filed on Feb. 2, 1990, now abandoned.

DE-A-2,939,269 (U.S. Pat. No. 4,226,941) and DE-A-2,717,440 (U.S. Pat.No. 4,168,963) disclose that the herbicidal action of racemicphosphinothricin or its salts with organic or inorganic bases or acidsoriginates from L-2-amino-4-methylphosphinobutyric acid (namedL-phosphinothricin or L-PTC in what follows) or salts thereof. TheD-form is virtually inactive. In contrast to the readily accessibleracemate of phosphinothricin, L-PTC could hitherto only be obtained bycomparatively complicated processes. There seemed to be a need todevelop a useful process by which the L-form can be made accessible inan economical way.

It has already been disclosed that L-PTC can be obtained by acidhydrolysis (JP-A-73-85538) or by enzymatic degradation (JP-A-74-31890)of L-PTC-alanyl-alanine, an antibiotic which is known from theliterature and obtained by microbial synthesis.

Furthermore, processes are known which are based on the enzymaticresolution of racemates of chemically synthesized racemic PTCprecursors. DE-A 2,939,269 describes a process in which N-acyl-PTC, inparticular N-acetyl-PTC, is cleaved with the aid of acylases which canbe obtained by specifically bred strains of microorganisms of the genusPseudomonas, Streptomyces or Aspergillus. In this process, N-acyl-L-PTCis cleaved more rapidly than N-acyl-D-PTC. According to the informationgiven by DE-A 2,939,269, the acylases used have hardly any, or only verylittle, effect on substrates other than N-acyl-L-PTC, for example onN-acyl derivatives of customary L-amino acids.

In contrast, DE-A-3,048,612 (U.S. Pat. No. 4,389,488) claims thatcommercially available acylases are usually not successful at theattempt to cleave rac-acyl-PTC. In this context, DE-A-3,048,612describes an advantageous individual case, according to which theactivity and selectivity of penicillin-G-acylase (pen-G-acylase) isimproved when phenacetyl-PTC is used. This was also surprising insofaras, according to A. Plaskie, J. Antibiotics 31, 783 (1978), it wasexpected that a variation on the amino acid moiety of the substrate incomparison with phenacetylated simple natural amino acids, such asphenacetylalanine or phenacetylvalin, would result in a sharp decreasein the pen-G-acylase activity or selectivity for deacylation of theL-isomer.

DE-A 2,927,534 (U.S. Pat. No. 4,389,489) or DE-A 2,215,853 (U.S. Pat.No. 3,813,317) discloses the resolutions of racemates of natural andsynthetic amino acids, in particular aryl-substituted amino acids, bymeans of carboxylic ester cleavage with the aid of proteolytic enzymes,in which case, again, it is the L-enantiomer which is preferentiallyreacted enzymatically. However, it should be noticed that no mention ismade in any of the cited publications that it might be possible tocleave amino acids having a phosphorus-containing radical. This isprobably due to the fact that P(V)-containing compounds, in particularderivatives of the general formula R'R"P(O)OR"' simulate thetransitional state of an enzymatically hydrolyzed carboxylic esterbecause of their sterical arrangement (see M. Dixon, E. Webb in"Enzymes" e. Edition, Longmans, Grenn & Co. LTD, London 1964, P.346-352) and, accordingly, could have a deactivating influence onhydrolytically active enzymes. In fact, DE-A 3,048,612 did indeeddescribe the fact that proteolytic enzymes which have esterase activityand which are highly active and selective in the case of conventionalD,L-amino acids, have no, or only a greatly reduced, activity in thecase of D,L-PTC esters.

On the basis of the prior art which has been mentioned, it could nothave been predicted that an effective enzymatic resolution of racematescould be carried out using readily accessible, diprotected ortriprotected PTC derivatives which are modified on the phosphinic acidmoiety of the PTC.

The invention relates to a process for the enzymatic resolution of PTCderivatives, which comprises treating a mixture of D- and L-PTCderivatives of the general formula (I) ##STR1## in which a) R¹ isunbranched or branched C₁ -C₂₀ -alkyl which is unsubstituted orsubstituted by one or more halogen radicals, such as fluorine, chlorine,bromine or iodine, or monosubstituted or poly-substituted by C₁ -C₈-alkoxy, or R¹ is C₃ -C₈ -cyclo-alkyl which can be substituted by one ormore groups from the series comprising C₁ -C₄ -alkyl, C₁ -C₄ -alkoxy andhalogen, or R¹ is C₃ -C₁₀ -alkenyl, C₃ -C₁₀ -alkynyl or benzyl,

R² is hydroxyl, unbranched or branched C₁ -C₂₀ -alkoxy which isunsubstituted or substituted by one or more halogen radicals, such asfluorine, chlorine, bromine and iodine, or monosubstituted orpolysubstituted by C₁ -C₈ -alkoxy, or R² is amino or (C₁ -C₂₀-alkyl)amino, and

R³ is formyl, unbranched or branched (C₁ -C₂₀ -alkyl)-carbonyl which isunsubstituted or substituted in the alkyl moiety by one or more radicalsfrom the series comprising hydroxyl, halogen, C₁ -C₄ -alkoxy, C₁ -C₄-alkylthio, and phenyl which can be substituted by up to three radicalsfrom the group comprising C₁ -C₁₂ -alkyl, C₁ -C₁₂ -alkoxy, halogen,nitro and CF₃, or R³ is benzoyl or benzoyl which is substituted by 1 to3 radicals from the group comprising C₁ -C₁₂ -alkyl, C₁ -C₁₂ -alkoxy,halogen, nitro and CF₃, or

b) R¹ is as defined for a),

R² is as defined for a), but is not hydroxyl, and

R³ is as defined for a), or is another protective group customary in thecase of amino groups, in particular selected from amongst unbranched orbranched (C₁ -C₂₀ -alkoxy)carbonyl and C₁ -C₂₀ -alkyl-sulfonyl, each ofwhich is unsubstituted or substituted in the alkyl moiety by one or moreradicals from the group comprising hydroxyl, halogen, C₁ -C₄ -alkoxy, C₁-C₄ -alkylthio, phenyl, phenyl which has 1 to 3 substituents selectedfrom the group comprising C₁ -C₁₂ -alkyl, C₁ -C₁₂ -alkoxy, halogen,nitro and CF₃, and phenylsulfonyl which can be substituted by up tothree radicals from amongst C₁ -C₁₂ -alkyl, C₁ -C₁₂ -alkoxy, halogen,nitro and CF₃,

with a hydrolytically active enzyme in an aqueous or aqueous-organicmedium, where, in case a), it is preferred to use an enzyme whichcleaves N-acyl groups (acylase) and, in case b), it is preferred to usea proteolytic or esterase-active enzyme.

A process according to the invention is of particular interest, and itcomprises using a mixture of D- and L-PTC derivatives of the indicatedformula (I), in which

a) R¹ is unbranched or branched C₁ -C₁₀ -alkyl or C₁ -C₁₀ -alkyl whichis substituted by halogen, such as fluorine or chlorine, or by C₁ -C₄-alkoxy, or R₁ is C₅ -C₆ -cycloalkyl,

R² is hydroxyl, unbranched or branched C₁ -C₈ -alkoxy or C₁ -C₈ -alkoxywhich is monosubstituted or polysubstituted by halogen, such as fluorineand chlorine, or by C₁ -C₄ -alkoxy, or R² is amino or (C₁ -C₁₀)-alkylamino, and

R³ is formyl, unbranched or branched (C₁ -C₁₀ -alkyl)-carbonyl which isunsubstituted or substituted in the alkyl moiety by one or two radicalsfrom the group comprising hydroxyl, halogen, phenyl, C₁ -C₄ -alkoxy, C₁-C₄ -alkylthio, and a phenyl radical which is substituted by 1 to 3radicals selected from amongst C₁ -C₄ -alkyl, C₁ -C₄ -alkoxy andhalogen, or R³ is benzoyl or benzoyl which is substituted by 1 to 3radicals selected from amongst C₁ -C₄ -alkyl, C₁ -C₄ -alkoxy andhalogen, or

b) R₁ is as defined for a),

R² is as defined for a), but is not hydroxyl, and

R³ is as defined for a) or is (C₁ -C₁₀ -alkoxy)carbonyl which isunsubstituted or substituted by hydroxyl, halogen, methoxy, ethoxy,phenyl, or a phenyl radical which carries one to three substituents fromthe group comprising C₁ -C₄ -alkyl, C₁ -C₄ -alkoxy and halogen.

A process according to the invention which comprises using a mixture ofD- and L-PTC derivatives of the indicated formula (I), in which

a) R¹ is unbranched or branched C₁ -C₆ -alkyl or is cyclohexyl,

R² is hydroxyl, unbranched or branched C₁ -C₆ -alkoxy, preferablymethoxy or ethoxy, or amino, and

R³ is (C₁ -C₄ -alkyl)carbonyl, preferably acetyl, or (C₁ -C₄-alkyl)carbonyl which is substituted by phenyl or by phenyl which ismonosubstituted to trisubstituted and whose 1 to 3 substituents areselected from amongst C₁ -C₄ -alkyl, C₁ -C₄ -alkoxy and halogen,preferably phenacetyl, or R³ is benzoyl, or

b) R¹ is unbranched or branched C₁ -C₆ -alkyl or is cyclohexyl,

R² is unbranched or branched C₁ -C₆ -alkoxy, or amino, and

R³ is as defined for a) or is unbranched or branched (C₁ -C₄-alkoxy)carbonyl, preferably tert.-butylo-xycarbonyl, or isbenzyloxycarbonyl which can be additionally substituted in the phenylring by up to three radicals from the group comprising C₁ -C₄ -alkyl, C₁-C₄ -alkoxy and halogen, is preferred.

C₁ -C₆ -alkyl is, in particular, methyl, ethyl, 1-propyl or 2-propyl,n-, i-, tert.- or 2-butyl, 3-methyl-2-butyl, n-, i-, tert.-, 2- or3-pentyl, n-hexyl or a stereo-isomeric hexyl.

C₁ -C₆ -alkoxy is, in particular, (C₁ -C₆ -alkyl)oxy, where the alkylradical in this case has the abovementioned meaning.

Unless defined in greater detail, halogen radicals are the radicalsfluorine, chlorine, bromine and iodine, preferably fluorine andchlorine, in particular chlorine.

The resolution according to the invention can be brought about by N-acylcleavage of the D,L-PTC derivatives of the formula (I) in which R¹, R²and R³ have the meanings given for a), or by hydrolytic enzymaticcarboxyl ester cleavage or carboxamide cleavage of the correspondingD,L-PTC derivatives of the formula (I) in which R¹, R² and R³ have themeanings given for b).

The N-acyl cleavage of the acylated α-amino group gives thecorresponding L-PTC derivative where R³ is H, which can be isolated fromthe aqueous solution of the reaction mixture in a customary manner, forexample by removing the unreacted D-PTC derivative and the acid whichhas been cleaved off, of the formula R³ --OH, by extraction at a pH inthe acid range with the aid of an organic solvent, in which process theL-PTC derivative remains in the aqueous solution in the form of theammonium salt and can subsequently be isolated by evaporating theaqueous solution to dryness. Furthermore, a separation bycrystallization, distillation, chromatography etc. is feasible.Alternatively, it is also possible to subject the substance mixture toalkaline hydrolysis at room temperature, for example at a pH of 12 orhigher. The optical antipodes of phosphinothricin are then present inthe form of free L-PTC (where R³ =H) or of N-acyl-D-PTC, and they can bepurified as described (cf. DE-A 2,939,269 and DE-A 3,048,612 which havebeen mentioned).

Suitable acylases for the process according to the invention with N-acylcleavage are those which have a sufficiently high hydrolytic activity inan aqueous or aqueous-organic medium. Such enzymes can be selectedeasily in preliminary experiments from the group of the customaryacylases.

Suitable enzymes for the N-acyl cleavage according to the invention ofphosphinic-ester-protected derivatives are, for example, acylase I (EC3.5.1.14), in particular for N-acetyl derivatives, andpenicillin-G-acylase (EC 3.5.1.11), in particular for phenacetylderivatives. Penicillin-G-acylase sometimes has other names in thespecialist literature, such as, for example, penicillin-G-amidase orpenicillin-amidohydrolase.

Alternatively, it is possible to cleave the carboxylic ester function orcarboxamide function enzymatically. In this case, the protected aminogroup in the α-position to the --CO--O-- or --CO--NH-- group can beprotected by almost any desired N-protective group, preferably by thecustomary N-protective groups as are described in Th. Greene, ProtectiveGroups in Organic Synthesis, John Wiley & Sons, New York 1981, inparticular on p. 218 et seq.

Suitable enzymes for the last-mentioned case are, in particular,proteolytic or esterase-active enzymes, in particular serineproteasesand thiolproteases, preferably subtilisin (EC 3.4.4.16, newclassification No. EC 3.4.21.4), α-chymotrypsin (EC 2.4.4.5, new class.No. 3.4.21.1), papain (EC 3.4.4.10, new class No. EC 3.4.22.2), ficin(EC 3.4.22.3) or bromelain (EC 3.4.22.4) the first two having a serineradical in the active center of the amino acid chain while the latterones have cystein as an active center of the amino acid chain.Subtilisin is preferred. Likewise, technical-grade enzyme qualities canbe employed as are known, for example, under the tradenames Maxatase®(manufacturer: Gist Borcades N.V., Delft/Netherlands) and Alcalase®(manufacturer: Novo Industrie AS, Copenhagen/Denmark).

The enzymatically hydrolyzed L-PTC derivative where R² is OH can besuccessfully isolated in a customary manner, or a manner known inprinciple, such as, for example, by crystallization, distillation,column chromatography or ion-exchange chromatography and, in particular,by rapid extraction, if appropriate at a reduced temperature of 0° to10° C., of the unreacted D-PTC derivative from the aqueous phase (thepreferred pH being 6-9) with the aid of a suitable organic solvent, inwhich process the L-PTC derivative resulting from a carboxylic estercleavage or carboxamide cleavage remains in the aqueous phase in theform of the carboxylate salt (R² =O-M+). In this process, the pH is tobe chosen in such a way that chemical hydrolysis is not yet initiated.After the D-derivative has been separated off, the L-PTC derivative issubjected to chemical hydrolysis analogously to customary methods, forexample using an aqueous dilute mineral acid at a temperature of 20° C.to the boiling point of the solution at reaction times of 1 to 24 hours.A suitable mineral acid is, for example, a hydrohalic acid or sulfuricacid, in particular dilute to concentrated hydrochloric acid. Inindividual cases, organic acids are also well suited. Total chemicalhydrolysis gives in addition to L-PTC also the carboxylic acid whichcorresponds to the acyl radical R³, it being possible to remove thecarboxylic acid from the aqueous-acid solution by distillation orextraction using a suitable organic solvent. When the aqueous phase isthen evaporated to dryness, L-PTC remains in the form of the ammoniumsalt.

The D-PTC derivatives which remain can optionally be subjected tofurther purification by extraction processes, distillation,crystallization or chromatography, and may then be purified of theby-products of the enzymatic hydrolysis, and, if appropriate afterracemization, for example by thermal means or by the action of a base,for example, an alcoholate in alcoholic solution, can be re-subjected toenzymatic hydrolysis as a D,L-mixture. This racemization with the baseusually proceeds under conditions which are particularly mild on thesubstance.

Said enzymes, in free form or after immobilization, can be employed inprocesses which are customary or known to the expert. The specificsubstrate is employed in the form of a solution or suspension in aqueousmedium. Possible concentrations are from 0.1% up to a saturated solution(e.g. the latter case if the process is carried out in suspension). Inindividual cases, it is possible to add water-soluble solvents, such asdimethylsulfoxide or methanol, and also to carry out the process in atwo-phase mixture with the addition of water-insoluble organic solvents.

In general, the reaction temperature for the enzymatic cleavages is10°-60° C., preferably 20°-50° C., in particular 20°-40° C. The processcan be carried out for example batchwise or continuously as a columnprocess.

Enzymatic hydrolysis is preferably carried out at a pH of 5 to 9, inparticular pH 6 to 8.5, it being also possible to carry out the processin individual cases at pH 5 to 6 in order to suppress unspecifichydrolysis of the carboxylic ester or carboxamide.

The procedure of the reaction can be monitored via the decrease of thesubstrate, for example HPLC. In individual cases, in particular in thecarboxylic ester cleavage, it is possible to determine the procedure ofthe reaction by other simple methods, for example by the amount of basewhich must be metered in to reach a constant pH.

The content of L-compound in the product of the enzymatic cleavage canbe determined with the aid of HPLC after alkaline or acid hydrolysis togive L-PTC has been carried out, followed by derivatization by a mannerknown per se (D. Aswad, Analytical Biochemistry 137, 405-409 (1984)).

The starting substances of the general formula I, with the exception ofthose where R² is amino or alkylamino, are known or can be prepared byprocesses known per se (cf. JP-75-7 237; WO 79-1114; JP 73-91019; MeijiSeika Kenkyo Nempo 1981, (20) 33-8). Individual examples in this contextare listed in the experimental part. Starting substances of the formula(I) in which R² is amino or alkylamino, are novel and can be prepared byselective hydrolysis and, if appropriate, N-alkylation, via the nitrilewhich corresponds to the carboxylic amide. The nitrites mentioned can beprepared, for example, as described in EP-A-194,521 (U.S. Pat. No.4,692,541). The invention therefore also relates to these novelcompounds, including the pure enantiomers and diastereomers thereofwhich come under the formula, and including mixtures of stereo-isomers.

The designation D or L in the optionally derivatized PTC derivativesonly indicates the configuration on the carbon atom which is in theα-position relative to the carboxylic group and which is linked to theoptionally protected amino group. Because of the additional center ofchirality on the phosphorus atom in the PTC derivatives protected withR¹, these D- or L-PTC derivatives are usually not pure enantiomers, butmixtures of diastereomers. As has emerged surprisingly, it isessentially only the configuration on the α-carbon atom mentioned whichis important for the enzymatic cleavage. After hydrolysis of the radicalR¹, the center of chirality on the phosphorus atom is virtually lostbecause of the rapid exchange of protons between OH and O.

The process according to the invention for the resolution of D,L-PTCderivatives and for the preparation of L-PTC is distinguished by aneffective resolution of substrate and product mixture, combined with alow amount of salt obtained as a by-product. If not desired, the D-PTCderivative which is obtained after the enzymatic resolution can beracemized readily and under mild conditions, if appropriate withoutprevious isolation, and can therefore be used again for an enzymaticresolution.

The fact there is a possibility to use commercially available acylasesfor the enantioselective cleavage of the PTC derivatives when those PTCderivatives are employed which have an ester-protected P-O acid functionaccording to the invention, is a particular, unexpected advantage. It issurprising that inhibition of the hydrolytic activity by the phosphinicester, or even the secondary reaction, hydrolysis of the phosphinicester, is generally not observed. The protection of the phosphinic acidas an ester has a positive impact on the reaction rate, in particular inthe case of the enzymatic carboxylic ester cleavage, or, in individualcases, makes acceptance of the substrate by the enzyme possible in thefirst place.

Moreover, it has emerged that, in spite of the massive changes of thestructural as well as chemical properties of a PTC derivative of theformula (I) in which R³ is phenacetyl and which is employed according tothe invention, compared with PTC which is only protected by oneN-phenacetyl group, as is used in accordance with DE-A-3,048,612, aneffective N-phenacetyl cleavage with pen-G-acylase combined with areaction rate which is at least identical and in some cases increased,is likewise observed. Because of the advantages which have beenmentioned for work-up, the process according to the invention is alsosuperior in this case.

A) Starting materials

EXAMPLE A1

Ethyl D,L-(3-phenylacetamido-3-ethoxycarbonylpropyl)-methylphosphinate(Formula I: R¹ =C₂ H₅, R² =OC₂ H₅, R³ =C₆ H₅ --CH₂ CO): 8.97 g (0.03mol) of D,L-(3-phenylacetamido-3-carboxy-propyl) methylphosphinicacid--prepared in accordance with JP-55-0025 (1980) or EP-A-054,897--aredissolved in a mixture of 10 ml of glacial acetic acid and 80 ml oftriethyl orthoformate, and the solution is refluxed for 3 hours. Thereaction mixture is rotated on a rotary evaporator and purified on asilica gel column (mobile phase CH₂ Cl₂ /methanol, 9:1). This gives 9.7g (91% of theory) of a colorless resin.

¹ H-NMR (CDCl₃): δ=7.4 (C₆ H₅,s,5H); 6.7 (NH,t,1H); 4.65 (CH,m,1H);3.55-4,2 (2×CH₂ CH₃,m,4H); 3.65 (CH₂ ;s,2H); 1.1-2.2 (PCH₃,CH₂ CH₂,2×CH₃ CH₂,m,13 H).

EXAMPLE A2

MethylD,L-(3-phenylacetamido-3-n-butoxycarbonylpropyl)-methylphosphinate(Formula I: R¹ =CH₃, R² =n--O--C₄ H₉, R³ =C₆ H₅ --CH₂ CO--):

a) D,L-(3-phenylacetamido-3-n-butoxycarbonylpropyl)-methylphosphinicacid:

50.0 g (0.167 mol) of D,L-(3-phenylacetamido-3-carboxypropyl)methylphosphinic acid are dissolved in 150 ml of n-butanol, onespatula-tip full of p-toluene sulfonic acid is added, and the mixture isthen refluxed for 5 hours. The reaction mixture is subsequentlyevaporated and the residue is crystallized by trituration withn-heptane. This gives 50 g (84% of theory) of colorless crystals of amelting point of 90° C.

b) Methyl D,L-(3-phenylacetamido-3-n-butoxycarbonylpropyl)methylphosphinate:

10.0 g (0.28 mol) of the product obtained in A2a) are reactedanalogously to Example 1 with a mixture of 10 ml of glacial aceticacid/80 ml of trimethyl orthoformate. This gives 6.0 g (58.2% of theory)of a colorless resin.

¹ H-NMR (CDCl₃): δ=7.2 (C₆ H₅,m,5H); 6.8 (NH,m,1H); 4.65 (CH,m,1H); 4.05(CH₂ CH₂ CH₃,t,2H); 3.8 (POCH₃,dd,3H); 3.7 (CH₂,s,2H), 0.8-2.2 (CH₂ CH₂CH₂ CH₃, PCH₃, CH₂ CH₂,m,14H).

EXAMPLE A3

Ethyl D,L-(3-phenylacetamido-3-carboxypropyl ) methylphosphinate

a) D,L-(3-Phenylacetamido-3-benzyloxycarbonylpropyl)-methylphosphinicacid:

29.9 g (0.1 mol) of D,L-(3-phenylacetamido-3-carboxy) methylphosphinicacid are suspended in 150 ml of methanol at room temperature, and thesuspension is treated with 19.2 g (0.106 mol) of tetramethylammoniumhydroxide pentahydrate. The solution is evaporated on a rotaryevaporator, the residue is dissolved in 250 ml of absolute DMF, and themixture is treated with 19.3 g (0.113 mol) of benzyl bromide at 0° C.After the reaction mixture has been stirred for 18 hours at roomtemperature, it is stirred into 600 ml of ice-water, and the mixture isextracted several times using CH₂ Cl₂ ; the CH₂ Cl₂ extracts aresubsequently dried over sodium sulfate and evaporated on a rotaryevaporator, and the crude product is freed from solvent residues at 50°C. under a high vacuum. This gives 28.6 g (73.5%) of a colorless oilwhich is employed in the next step without further purification.

b) Ethyl D,L-(3-phenylacetamido-3-benzyloxycarbonylpropyl)methylphosphinate:

20.0 g (0.05 mol) ofD,L-(3-phenylacetamido-3-benzyloxycarbonylpropyl)methylphosphinic acidare refluxed for 3 hours with 50 ml of glacial acetic acid and 150 ml oftriethyl orthoformate. The reaction mixture is evaporated on a rotaryevaporator, and the product is purified by chromatography on silica gel(mobile phase: ethyl acetate/methanol (8:1)). This gives 11.9 g (56.7%of theory) of the desired product which is employed in the next stepwithout further purification.

c) Ethyl D,L-(3-phenylacetamido-3-carboxypropyl)methylphosphinate:

11.0 g (0.026 mol) of ethyl D,L-(3-phenylacetamido-3-benzyloxycarbonylpropyl)methylphosphinate aredissolved in 250 ml of ethanol, 5 g of palladium on activated carbon (5%of substance) are added, and the mixture is then hydrogenated underatmospheric pressure. After 2 hours, the catalyst is filtered off andthe filtrate is evaporated on a rotary evaporator, and the residue istriturated with n-heptane. This gives 6.2 g (72.9% of theory) ofcolorless crystals of m.p. 112°-117° C.

EXAMPLE A4

Methyl D,L-(3-acetamido-3-methoxycarbonylpropyl)methylphosphinate:

10 g (0.05 mol) of the ammonium salt of D,L-(3-amino-3-carboxypropyl)methylphosphinic acid are suspended in 40 ml of glacialacetic acid, 50 ml of 1,1,1-trimethoxyethane are added, and the mixtureis then refluxed for 3 hours. It is subsequently filtered, and thefiltrate is concentrated and chromatographed on silica gel (mobile phaseCH₂ Cl₂). This gives 8.75 g (75% of theory) of the desired product inthe form a yellowish oil; ¹ H-NMR (CDCl₃): δ=7.18 (NH,m,1H); 4.6(CH,m,1H); 3.75 (COOCH₃, s,3H); 3.7 (POCH₃,dd,3H), 1.5-2.2 (CH₂CH₂,m,4H); 2.05 (CH₃ CO--,s,3H); 1.45 (PCH₃,d,3H).

EXAMPLE A5

Ethyl D,L-(3-acetamido-3-ethoxycarbonylpropyl)methylphosphinate:

10 g (0.05 mol) of the ammonium salt of D,L-(3-acetamino-3-carboxypropyl)methylphosphinic acid are reacted analogously to ExampleA4, but using glacial acetic acid/triethyl orthoformate. This gives 8 g(57.6% of theory) of the desired product in the form of a colorless oil.

¹ H-NMR (CDCl₃): δ=6.95 (NH,m,1H); 4.6 (CH,m,1H); 4.24 (COOCH₂CH₃,q,3H); 4.08 (POCH₂ CH₃,m,3H); 2.07 (CH₃ CO,s,3H); 1.8-2.2 (CH₂CH₂,m,4H); 1.47 (PCH₃,d,3H); 1.2-1.45 (POCH₂ CH₃,m,3H).

EXAMPLE A6

CyclohexylD,L-(3-phenylacetamido-3-aminocarbonylpropyl)-methylphosphinate

a) Cyclohexyl D,L-(3-phenylacetamido-3-cyanopropyl)-methylphosphinate:

28.7 g (0.1 mol) of cyclohexylD,L-(3-acetoxy-3-cyanopropyl)methylphosphinate (prepared analogously toEP-A-127,577) are added dropwise at 20° C. within 1 hour to 29.6 ml ofconcentrated ammonia. The reaction mixture is subsequently extractedusing CH₂ Cl₂, and the CH₂ Cl₂ extract is dried over sodium sulfate andtreated with 10.2 g (0.1 mol) of triethylamine. 15.4 g (0.1 mol) ofphenylacetyl chloride are added dropwise at 0° C. After the mixture hasbeen stirred for 18 hours at room temperature, it is treated with 50 mlof water, a pH of 5 is established using 0.5N hydrochloric acid, and themixture is extracted using CH₂ Cl₂. The oil which remains after the CH₂Cl₂ extract has been evaporated is purified by chromatography on silicagel (mobile phase CH₂ Cl₂). This gives 27.5 g (76% of theory) of thedesired product;

¹ H-NMR (CDCl₃): δ=9.45 (NH,m,1H); 7.3 (C₆ H₅ s,5H); 4.95 (CH,m,1H);4.36 (CH,m,1H); 3.6 (CH₂,s,2H); 1.2-2.2 (CH₂ CH₂,PCH₃,C₆ H₁₀,m,17H).

b) CyclohexylD,L-(3-phenylacetamido-3-aminocarbonylpropyl)methylphosphinate:

6 g (0.0165 mol) of cyclohexyl D,L-(3-phenylacetamido-3-cyanopropyl)methylphosphinate are dissolved in 40 mlof formic acid, and HCl gas is subsequently passed in at roomtemperature. After 3 hours, the reaction mixture is evaporated, theresidue is dissolved in methylene chloride and water, and a pH of 5 isestablished using sodium hydrogen carbonate.

The mixture is subsequently extracted using CH₂ Cl₂, and the CH₂ Cl₂extracts are dried over Na₂ SO₄ and evaporated on a rotary evaporator.The crude product which remains is purified by chromatography on silicagel (mobile phase CH₂ Cl₂ /MeOH, 9:1). This gives 4.18 g (66.7% oftheory) of a pale yellow oil;

¹ H-NMR (CDCl₃): δ=7.2 (C₆ H₅,s,5H); 7.2 (CONH₂,d,2H);

5.6 (NH,s,1H); 4.2-4.8 (2×CH,m,2H); 3.6 (CH₂,s,2H);

1.2-2.3 (CH₂ CH₂,PCH₃,C₆ h₁₀,m,17H).

B) Enzymatic enantio-differentiating hydrolyses

EXAMPLE

    ______________________________________     ##STR2##                 R.sup.1   R.sup.2    R.sup.3    ______________________________________    (1)          Et        OH         Phac    (2)          Et        OH         H    PTC          H         OH         H    ______________________________________

1.2 g of ethyl D,L-(3-phenylacetamido-3-carboxypropyl)-methylphosphinate(D,L-(1)) are taken up in 40 ml of H₂ O, and, after the pH has beenadjusted to 7.8, the mixture is stirred with 0.2N aqueous ammoniasolution in the presence of 2 ml of fixed penicillin-G-acylase (66units/ml) at 35° C. The pH is kept constant by adding 0.2N aqueousammonia solution. After 2 hours, the enzyme is filtered off, a pH of 2.5is established using concentrated hydrochloric acid, and the remainingsubstrate D-(1) and phenyl acetic acid are rapidly extractedexhaustively using methyl isobutyl ketone. Evaporation of the aqueousphase to dryness under reduced pressure gives 550 mg of ethylL-(3-amino-3-carboxypropyl)methylphosphinate hydrochloride (L-(2)),containing NH₄ Cl as impurity; a!_(D) ²² =+8.3° (c=5 in H₂ O), ¹ H-NMR(D₂₀) of L-(2): δ=3.9-4.3 (CH₃ CH₂ O, CH-NH₂,m, 3H); 1.8-2.4 (CH₂ CH₂,m, 4H); 1.65 (P-CH₃, d, 3H); 1.3 (CH₃ CH₂ O, t, 3H).

To determine the selectivity of the enzymatic cleavage, theL-(2)-hydrochloride is first refluxed for 6 hours in 20 ml ofconcentrated hydrochloric acid and the product is evaporated to dryness,i.e. chemically hydrolyzed. A 10 mg sample is then dissolved in 10 ml ofa buffer solution at pH 10, the solution is treated with 134 μl of asolution of 1 g of BOC-cysteine in ethanol together with 67 μl of asolution of 333 mg of o-phthalic dialdehyde in 5 ml of ethanol, and,after 10 minutes, the mixture is analyzed via HPLC (column: LiChrosorbRP-18; mobile phase: 50 mmol phosphate buffer/methanol/tetrahydrofuran:71/28/1); R_(t) (L-PTC)=6.0 minutes; R_(t) (D-PTC)=6.6 minutes. Theproportion of pure L-(2)-hydrochloride after the enzymatic cleavage isthus calculated indirectly via the L-PTC as at least 94%.

EXAMPLE

    ______________________________________     ##STR3##                 R.sup.1   R.sup.2    R.sup.3    ______________________________________    (3)          Et        OEt        Phac    (1)          Et        OH         Phac    ______________________________________

200 mg of ethylD,L-(3-phenylacetamido-3-ethoxycarbonylpropyl)methylphosphinate(D,L-(3)) are suspended in 40 ml of H₂ O, and a pH of 8 is establishedusing 0.2N aqueous ammonia solution. After 10 mg of subtilisine havebeen added, the mixture is allowed to react for 2 hours at 35° C. D-(3)is extracted exhaustively from the solution using methyl isobutylketone. The aqueous phase is adjusted to pH 2 using concentratedhydrochloric acid and likewise extracted exhaustively using methylisobutyl ketone. Evaporation to dryness of the aqueous phase gives 75 mgof ethyl L-(3-phenylacetamido-3-carboxypropyl)-methylphosphinate (L-(1))having an optical rotation of α!_(D) ²² =+25° (4% in CHCl₃); ¹ H-NMR (D₂O): δ=7.3 (phenyl, 5H); 4.6-4.3 (CH--NH, m, 1H); 4.3-3.7 (CH₃ CH₂ O, m,2H); 3.65 (CH₂ C₆ H₅, s, 2H); 2.4-1.5 (CH₂ CH₂, m, 4H); 1.5 (P--CH₃, d,3H); 1.25 (CH₃ CH₂ O, t, 3H).

L-(1) is taken up in concentrated hydrochloric acid, and the mixture isrefluxed for 6 hours and subsequently evaporated to dryness. Theproportion of L-PTC is determined via HPLC as described in Example B1:accordingly, the proportion of L-compound is at least 92%.

EXAMPLE

    ______________________________________     ##STR4##                 R.sup.1   R.sup.2    R.sup.3    ______________________________________    (3)          Et        OEt        Phac    (4)          Et        OEt        H    ______________________________________

2 g of ethylD,L-(3-phenylacetamido-3-ethoxycarbonylpropyl)methylphosphinate(D,L-(3)) are suspended in 40 ml of H₂ O, the pH is adjusted to 8, andthe mixture is stirred at 35° C. in the presence of 2 ml of fixedpenicillin-G-amidase (132 units). The pH is kept constant by adding 0.2Naqueous ammonia solution. After 2 hours, the enzyme is filtered off, thereaction solution is adjusted to pH 2 using concentrated hydrochloricacid, and unreacted substrate D-(3) and phenylacetic acid are extractedexhaustively using methyl isobutyl ketone. The aqueous phase isevaporated to dryness in vacuo, this giving 729 mg of ethylL-(3-amino-3-ethoxycarbonyl)methylphosphinate (L-(4)) having an opticalrotation of a!_(D) ²² =+15° (c=5 in H₂ O), containing impurities of NH₄Cl; ¹ H-NMR (D₂ O): δ4.5-3.75 (2×CH₃ CH₂ O, CHNH₂, m, 5H); 2.4-1.75 (CH₂CH₂, m, 4H); 1.6 (P--CH₃), d, 3H); 1.3+1.33 (2×CH₃ CH₂ O, t, 6H).

L-(4) is converted to L-PTC and analyzed as described in Example B1;accordingly, the proportion of pure L-(4) is 96%.

The substrate D-(3) which has been extracted and has not been reactedenzymatically is taken up in methyl isobutyl ketone, washed once using 1N NaOH and purified by column chromatography (CH₂ Cl₂ :MeOH 10:1).

Yield: 700 mg of D-(3) α!_(D) ²² =-9° (CHCl₃)

EXAMPLE B4

1.2 g (3.8 mmol) of methylD,L-(3-phenylacetamido-3-methoxycarbonylpropyl)methylphosphinate arereacted with 1.2 g of fixed penicillin-g-amidase (240 u) in 50 ml of0.01 M aqueous potassium phosphate buffer at pH 7. After 24 hours, theenzyme is filtered off, and the filtrate is adjusted to pH 3 andextracted exhaustively using methyl isobutyl ketone. The aqueous phaseis extracted to dryness, a pH of 1 is subsequently established by addingconcentrated hydrochloric acid, and the mixture is refluxed for 6 hours.Evaporation to dryness gives 420 mg of L-phosphinothricin; theproportion of L-PTC, determined via HPLC analogously to Example B1, is93%.

EXAMPLE B5

700 mg (2.95 mmol) of methylD,L-(3-acetamido-3-methoxycarbonylpropyl)methylphosphinate are taken upin 25 ml of water and, after the addition of 700 mg of acylase I, and0.37 ml of a 0.1M CoCl₂ solution, were stirred at room temperature.After 19 hours, a pH of 12 to 13 is established, and the mixture isstirred for 24 hours at room temperature. The solution is evaporated todryness and subsequently purified by ion-exchange chromatography on anacid ion exchanger (development with H₂ 0). The PTC-containing fractions(ninhydrin test, controlled by HPLC) were combined and evaporated todryness. This gives 250 mg of a brown powder which contains L-PTC in aproportion of more than 95% (determination via HPLC analogously toExample B1).

EXAMPLE B6

500 mg of optically pure ethylD-(3-phenylacetamido-3-ethoxycarbonylpropyl)methylphosphinate (D-(3)),originating from an enzymatic cleavage as described in Example B2, aretaken up in 15 ml of H₂ O, a pH of 8 is established, and the mixture isthen stirred at 35° C. with 2N NH₄ OH with 1 ml of penicillin-G-acylase(66 u). When the reaction is monitored via HPLC (column: LiChrosorbRP-8, Merck Hibar; mobile phase: 1 g of TBAHS+10 g of KH₂ PO₄ in 1 l ofH₂ O; pH 2.1 adjusted using H₃ PO₄, +1 l of methanol, UV detector 206nm), phenylacetic acid is no longer detectable after 48 hours.Accordingly, pen-G-acylase is only capable of splitting L-(3), but notD-(3).

EXAMPLE B7

500 mg of cyclohexylD,L-(3-phenylacetamido-3-aminocarbonylpropyl)methylphosphinate are takenup in 20 ml of 0.5M potassium phosphate buffer of pH 8, 2 ml (130 u) offixed pen-G-acylase are added, and the mixture is then stirred at 35° C.The pH is kept constant by continuously adding 0.2N NH₃ solution from aburette. After 18 hours, the enzyme is filtered off, and the filtrate isextracted exhaustively using methylene chloride. The aqueous phase isadjusted to pH 2 using concentrated hydrochloric acid, phenylacetic acidis extracted using methylene chloride, and the aqueous phase isevaporated to dryness. The residue is taken up in 20 ml of concentratedhydrochloric acid, and the solution is refluxed for 7 hours and thenagain evaporated to dryness.

The content of L-PTC is determined via HPLC as described in Example B1;proportion of L-PTC: 94%.

EXAMPLE B8

Racemization

100 mg of ethylD-(3-phenylacetamido-3-ethoxycarbonylpropyl)methylphosphinate (D-(3))having an optical rotation of α!_(D) ²⁰ =-9° (CHCl₃) are dissolved in 12ml of EtOH, and the solution is treated with 2 mg of sodium ethanolate.After 5 hours, the solution is evaporated to dryness. The opticalrotation of the product is α!²² =0° (CHCl₃)

We claim:
 1. A process for the enzymatic resolution of PTC derivatives,which comprises treating a mixture of D- and L-PTC derivatives of thegeneral formula (I) ##STR5## in which R¹ is unbranched or branched C₁-C₆ -alkyl or is cyclohexyl,R² is unbranched or branched C₁ -C₆ -alkoxy,or amino, and R³ is (C₁ -C₄ -alkyl)carbonyl, or (C₁ -C₄ -alkyl)carbonylwhich is substituted by phenyl or by phenyl which is monosubstituted ortrisubstituted and whose 1 to 3 substituents are selected from the groupconsisting of C₁ -C₄ -alkyl, C₁ -C₄ -alkoxy and halogen, or R³ isbenzoyl, or is unbranched or branched (C₁ -C₄ -alkoxy)carbonyl, or isbenzyloxycarbonyl which additionally can be substituted in the phenylring by up to three radicals selected from the group consisting of C₁-C₄ -alkyl, C₁ -C₄ -alkoxy and halogen, with a hydrolytically activeprotease enzyme in an aqueous or aqueous-organic medium.
 2. The processas claimed in claim 1, whereinR¹ is methyl, ethyl, propyl, butyl,pentyl, hexyl or cyclohexyl, R² is methoxy, ethoxy, propoxy, butoxy,pentoxy, hexoxy or NH₂, and R³ is acetyl or phenacetyl, or istert-butyloxycarbonyl or benzyloxycarbonyl.
 3. The process as claimed inclaim 1, wherein the enzymatic cleavage is carried out at a temperatureof 20° to 60° C.
 4. The process as claimed in claim 1, whereinenzymatically unreacted D-PTC derivative is removed after enzymaticcleavage by extraction with an organic solvent.
 5. The process asclaimed in claim 1, wherein L-PTC derivative obtained is subjected tochemical hydrolysis and L-PTC is isolated.
 6. The process as claimed inclaim 1, wherein the enzymatic cleavage is carried out at pH 5 to 9.